Our lab is studing a new class of mammalian cell mutants - mutants with defects in oxidative energy metabolism. These mutants have been classified into seven genetic complementation groups, three of which involve defects in Complex I of the electron transport chain. Experiments are described in this proposal to further characterize these respiration-deficient mutants with defects in Complex I. Experiments will be done to determine if all of the enzymatic activities associated with Complex I or requiring a functional Complex I (including NAD(P)H-ubiquinone reductase, NAD(P)H-ferricyanide reductase, NAD(P)H-NAD+ transhydrogenase, the energy-linked reduction of NAD+ and NADP by succinate and the oxidation of NADH by fumarate) are missing or altered in these mutants. Experiments will be done to determine if any of the peptides of Complex I are missing or altered in charge or molecular weight in these mutants. We will attempt to correlate the absence or alteration of a specific polypeptide with the absence or alteration of a specific enzymatic activity. EPR studies of mutant and wild-type mitochondria will be carried out to determine if any of the iron-sulfur centers of Complex I are absent in the mutants. Experiments will also be done to isolate and characterize additional mammalian cell mutants with defects in oxidative energy metabolism - including attitional mutants with defects in Complex I. A detailed biochemical and genetic analysis of a series of mammalian cell mutants with defects in Complex I should provide information about the structure, function and regulation of this important complex of the electron transport chain.